PROJECT SUMMARY/ABSTRACT The only curative treatment option for end-stage lung disease is transplantation, however, this therapy is plagued by limitations: those who are fortunate enough to receive a donor lung face a lifetime of immunosuppression and a ten-year survival rate of less than 30%. Nevertheless, evidence is accumulating for the innate potential of numerous epithelial and mesenchymal cell types in the lung to promote repair and regeneration after injury, even at the whole tissue level. This project proposes to use whole lung engineering strategies to investigate the role of epithelial-mesenchymal interactions, along with a key developmental signaling pathway, in promoting alveolar epithelial differentiation and alveolar unit formation. Specifically, the overarching goal of this proposal is to investigate whether alveolar epithelial type II cell-fibroblast interactions and canonical Wnt-?-catenin soluble effectors can be modulated in vitro in a 3-dimensional (3-D) biomimetic rat lung model to engineer a distal lung epithelium with improved surfactant production and epithelial barrier function. The first Aim of this project focuses on characterizing the pattern of canonical Wnt signaling in the native lung, as well as in isolated type II cells (here RTII+ cells, selected for by the rat type II marker RTII-70) and fibroblasts (FBs), during alveologenesis. Patterns of Wnt signaling during this late stage of lung development, during which mature type II cells produce surfactant, differentiate into type I cell progeny, and become part of the functional alveolus, will likely inform alveolar repair processes. The second Aim focuses on evaluating the effects of canonical Wnt soluble effectors on the phenotype and differentiation of RTII+ cells, with and without FB co-culture, using standard 2-D and 3-D organoid culture systems. It seeks to determine whether temporal modulation of these effectors, according to a timescale determined in Aim 1, might be utilized to promote maturation of alveolar epithelium in vitro. Finally, the third aim looks at the incremental effect of decellularized lung extracellular matrix on the maturation of RTII+ cells, with or without FB co-culture. In this last Aim, RTII+ cells and FBs will be seeded into an acellular rat lung scaffold and mounted into a biomimetic bioreactor. This system allows for assessment of RTII+ cell function at the whole tissue level, in terms of lung compliance and epithelial barrier function. Overall, this project has important implications, both for understanding the factors that promote the formation of a mature alveolar epithelium capable of gas exchange, as well as for the development of strategies to promote lung regeneration endogenously in patients suffering from chronic lung disease.